The overarching goal of this research program is to determine the molecular mechanism of both substrate and inhibitor recognition by lipoxygenase and apply this knowledge to understanding the role of lipoxygenase in cellular biology and human disease. Human lipoxygenase (hLO) isozymes are critical therapeutic targets because they are involved in numerous human diseases and yet, fundamental questions remain regarding their biochemistry and their role in cellular biology. We propose to investigate both the biochemical and biological properties of soybean 15-LO-1 and three hLOs, platelet 12- hLO, reticulocyte 15-hLO-1 and epidermal 15-hLO-2, using a multi-faceted approach, including kinetics, spectroscopy, calorimetry, crystallography, computer modeling, inhibitor screening and whole cell inhibitor assays. The first aim is to determine the manner in which LO binds substrate and how inhibitors and liposomes affect the substrate specificity. Lipoxygenases react with a variety of substrates, including arachidonic acid and linoleic acid, producing products with a wide range of functions but the manner in which the catalytic site differentially binds these substrates and converts them to products remains unclear. In this aim, we propose experiments which will define how the substrate is bound, what conditions change its substrate specificity and how LO obtains its substrate from the lipid bilayer. The second aim is to determine the solution structures of LO and how inhibitors and liposomes affect change. Our current structural understanding of LO is largely limited to a few static crystal structures that say nothing of how the substrate enters and docks to the catalytic site, or how LO recruits substrate from the lipid bilayer. In this aim, we shall utilize a variety of structural methods, such as proteolysis, H/D exchange, EPR spin labeling, and crystallography, to probe the structure of LO. Specifically, we will investigate whether the structures of the hLOs match that of the crystallized soybean and rabbit LOs, what structural changes occur upon substrate or inhibitor binding and how the LO-substrate-lipid complex interacts to achieve catalysis. The third aim is to utilize our discovered inhibitors to optimize our virtual screen, perfect human LO active site models, define the substrate specificity effect, and probe the cellular role of LO in human disease. We propose to utilize our previously discovered potent and selective inhibitors from our high throughput screen, to perfect our human structural model and improve our virtual docking. Extensive kinetic studies will be performed to assess the inhibitory mechanism of these compounds, be it allosteric or competitive or reductive. This family of specific inhibitors will then constitute a tool box which will allow us to probe the regulation of LO's specific activity via allosteric binding, and the role of LO in both prostate cancer and neuronal cell death (i.e. stroke). Lipoxygenase (LO) is a critical enzyme involved in numerous human diseases, such as cancer, stroke and heart disease. The goal of this application is to discover and characterize inhibitors to LO with the hope of learning more about its biochemical and cellular mechanism and developing possible therapeutics.